Chromosomal multidrug resistance (MDR) in bacteria is a serious clinical problem. Our studies have shown that Escherichia coli becomes resistant to a variety of antibiotics, organic solvents and superoxides when the activities of any of three paralogous, but differently regulated, transcriptional activators, MarA, SoxS and Rob, are increased. These activators bind a sequence called the marbox which lies upstream of the promoters of a set of at least 40 chromosomal genes called the marA/soxS/rob regulon. One of the genes upregulated by MarA, SoxS and Rob is tolC, which encodes a protein that is essential for multi-antibiotic resistance in E. coli. TolC is an outer membrane protein that interacts with nine different inner membrane efflux pumps and cognate periplasmic proteins to form a tripartite trans-periplasmic channel for extruding antibiotics directly out of the cell into the environment. We have recently inferred an additional function for TolC and the pumps: to expel toxic cellular metabolites. The nature of the pumps involved and the metabolites excreted is the subject of our current research. Previously, we found that E. coli tolC mutants, which do not have the TolC outer membrane channel, have elevated levels of transcription of marRAB and soxS and have elevated activity of Rob protein. Since TolC is a vital component of eight known efflux pumps in E. coli and plays important roles in ridding bacteria of multiple antibiotics, bile salts, organic solvents and other xenobiotics, we concluded (1) that in the absence of TolC, intracellular metabolic waste products accumulate and trigger the upregulation of the three activators and (2) that TolC is normally involved in the efflux of cellular metabolites and not merely of xenobiotics. We now find that three TolC-dependent efflux pumps are involved in regulating the concentration of these metabolite(s): AcrAB, EmrAB and MdtEF. This, and other data, show that the regulation of MarA, SoxS and Rob by TolC is due to specific kinds of efflux and not to an unknown function of TolC. We also show that two important stress-response systems, encoded by baeRS and cpxARP, are upregulated in tolC mutants. These two systems regulate a number of genes involved in maintaining periplasmic proteins in their properly folded configurations. One of the genes they regulate, spy, encodes a novel ATP-independent periplasmic chaperone. Using spy::lacZ transcriptional fusions, we have found 6- to 8-fold increases in transcription of spy in tolC mutants or in mutants defective for the following four TolC-dependent pumps: AcrAB, acrAD, MdtABCD and MdtEF. Why defective efflux increases Spy chaperone expression remains to be determined but we believe that in the absence of efflux certain metabolites accumulate and poison extracytoplasmic proteins whose folding may be corrected by Spy. In addition to regulation of BaeRS, CpxARP, MarA, SoxS and Rob by TolC, the important extracytoplasmic sigma factor Sigma E is regulated by TolC but whether this is due to efflux has not been ascertained (Corbaln et al., 2010). Furthermore, we previously showed that TolC plays a role in extreme acid survival (necessary for survival in the stomach) and that the TolC-dependent MdtABCD and EmrAB pumps are largely responsible for the poor survival. All this is strong evidence that the TolC efflux pump system of E. coli is very important in regulating the excretion of potentially toxic cellular metabolites. A better understanding of the TolC-dependent efflux pumps may help us to understand their function in antibiotic resistance and how antibiotic resistance may be overcome by countering these functions.